in- vitro and in-vivo screening of hypoglycemics
TRANSCRIPT
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In-vivo and In-vitro Screening of Hypoglycemics
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Presented by Kamlesh V. Warokar
M.Pharm (Semister-I) (Dept. of Pharmacology)
S.I.O.P., Pune.
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• Introduction & Type Of D.M.
• Symptoms of D.M.
• Classification of Hypoglycemic drugs.
• In-vivo screening of Hypoglycemics.
• In-vitro screening of Hypoglycemics.
• Evaluation Parameter.
• References.
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Contents
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INTRODUCTION
• Diabetes Mellitus(DM) is a metabolic disorder characterized by hyperglycemia, glycosuria, hyperlipidaemia, & ketonaemia.
• Hyperglycemia is a metabolic disorder characterized by high blood sugar level which may leads to diabetes mellitus.
• High blood glucose happen when the body has too little insulin or when body can not use insulin properly.
• On this basis diabetes mellitus classified as : Type I- Insulin Dependent Diabetes Mellitus(IDDM), Type II-Noninsulin Dependent Diabetes Mellitus
(NIDDM). 3
(K.D.Tripathi et al.)
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1. Juvenile onset of DM. 2. Beta cells destruction in
pancreatic islets. 3. Less common. 4. Have low degree of genetic
predisposition.
1. Maturity onset of DM. 2. Low insulin circulation &
moderate reduction in beta cell mass.
3. Most common. 4. High degree of genetic
predisposition.
TYPE 1 TYPE 2
(K.D.Tripathi et al.)
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SYMPTOMS OF HYPERGLYCEMIA
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Major Symptoms
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1. SULFONYLUREAS A. First generation- Tolbutamide, Chloropropamide.
B. Second generation- Glipizide, Gliclazide. 2. BIGUANIDES- Metformin.
3. MEGLITINIDE- Repaglinide, Nateglinide
4. THIAZOLIDINEDOINES- Pioglitazone.
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CLASSIFICATION OF HYPOGLYCEMIC DRUGS
(K.D.Tripathi, et al.)
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1. Streptozotocin induced diabetes2. Alloxan induced diabetes3. Pancreatectomy in dogs4. Other diabetogenic compounds5. Hormone induced diabetes6. Insulin deficiency due to insulin
antibodies7. Virus induced diabetes
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METHODS FOR SCREENING OF HYPOGLYCEMICS
1. In vivo models for IDDM
(Vogel,G.H.,et al.)
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A. Spontaneous or genetically derived diabetic animal.
B. Diet / Nutrition induced diabetic animal.
C. Neonatal STZ induced diabetic animal.
D. Transgenic diabetic animal.
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2. In vivo models for NIDDM
(K. Srinivasan & P. Ramarao)
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INVITRO SCREENING OF HYPOGLYCEMICS
1. Effect on Liver
2. Effect on Muscles
3. Effect on Pancreas
4. Effect on Adipose Tissue
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(Vogel,G.H.,et al.)
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ANIMALS USED
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1. STREPTOZOTOCIN INDUCED DIABETES
Male Wister Rats(150-220gm) are injected i.p. with 60mg/kg STZ prepared in citrated buffer.
Three phases of changed blood glucose level are observed.
Principle and Rationale
Procedure
IN VIVO MODELS FOR IDDM
Rakieten et al.(1963) discovered diabetogenic activity of the antibiotic Streptozotocin. Basic principle is compound is found to be cytotoxic to beta-cells of the pancreas.
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Initially after 3 hrs glucose level increased up to 150-200mg/dl . After 8 hrs, serum insuline values are incresed
up to 4 times. Hypoglycemic phase followed by hyperglycemia.
After 24-48 hours hyperglycemia already occur reaching values 800mg/dl with glycosuria & ketonemia.
Histologically beta cells are degranulated.
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After 14 days animal used for pharmacological test.
(Vogel,G.H.et al.)
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2. ALLOXAN INDUCED DIABETES
Purpose and Rationale
Frerichs(1968) & Creutzfeldt(1971)- Chemically induced diabetes in animals.
Procedure Animal is injected with a single dose [100 mg/kg body weight] dissolved in normal saline by i.p. route
Animals are kept for 48 hours during which food and water is allowed.
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Blood glucose levels show triphasic response with hyperglycemia for 1 hour followed by hypoglycemia that lasts
for 6 hours & stable hyperglycemia after 48 hours.
Animals showing fasting blood glucose level above 140 mg/dl after 48 hour are considered diabetic
For a period of six weeks, drug samples to be screened are administered orally
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After six weeks of treatment, blood samples are collected from 8 hour fasting animals (can be collected via orbita sinus through a pipette)
The serum glucose level is estimated by glucose oxidase-peroxidase method [GOD-POD] using autoanalyser.
(Vogel,G.H.,et al.)
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Animal used
Beagle dogs 60 mg/kg(i.v)
Wistar rat 100-175 mg/kg(s.c)
Rabbits 150mg/kg (via ear vein)
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Mechanism Of Action
(S. Lenzen.)
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3. PANCREATECTOMY IN DOGS
Principle or Rationale
Procedure
Dogs-(12-16kg)
Anesthesia- Phenobarbital sodium (50mg/kg).Remove fur and disinfect.
Incision- Xyphoid to Umbilicus.
Self retaining retractor applied. Pancreas is brought in operating field. Pancreas separated from duodenum and
dissected freely.
Diabetes can be achieved by removal of pancreas.Bomskov(1910)- severe diabetic symptoms in dogs.
Von Mehring & Minkowski(1890)- polyuria, polydipsia, after removal of pancreas.
(Vogel,G.H.,et al.)
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PANCREATECTOMY
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RETRACTOR
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• distal pancreatectomy surgery medicine surgical procedures.mp4
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4. OTHER DIABETOGENIC AGENTS
Purpose and Rationale
Procedure Chleators such as dithizone injected in rabbit at dose of 40-100mg/kg which leads to triphasic glycemic reaction.
Phase 1- Increased blood glucose level after 2hr.Phase 2- Hypoglycemic phase after 8hr.
Phase 3 – Permanent hyperglycemia after24-72hr.
Diabetogenic agents are Dithizone, Goldthioglucose or monosodiun glutamate. Rabbit, Cats, Hamster rat and mice can be employed for this model.
(Vogel,G.H.,et al.)
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5. HORMONE INDUCED DIABETESGrowth
hormone
Cotes et al., (1949) Animals used are generally adult Cats and Dogs.
Repeated administration of GH induce diabetes with the symptoms of ketonuria & ketonemia.
Rats does not shows diabetes but grow faster and shows striking hypertrophy of pancreatic islet.
(Karthikeyan M.,et al.)
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Ingle (1941) Forced fed rats, guinea pig and rabbits without
forced feeding are used.
In rats adrenal cortex is stimulated by corticotropin, which secret steroids and
induce steroid diabetes.
Dexamethasone is mostly used.
Corticosteroid
(Karthikeyan M.,et al.)
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Adult rats 150-200gm dexamethasone 2-5 mg/kg i.p.
Repeated injection of same dose level is carried out for a period of 20-30 days resulting in Diabetes.
The sample to be screened is administered through a suitable route, blood glucose is measured.
PROCEDURE
(Vogel,G.H.,et al.)
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6. VIRUS INDUCED DIABETES
Principle & Rationale Jevunile onset diabetes mellitus may be due
to virus infection and beta-cell specific autoimmunity. (Craighead 1978).
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6-8 week old mice are injected with D- variant of encephalomyocarditis [EMC] i.p.
Procedure
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Pre treatment is given with cyclosporin A.
Drug to be screened is administered orally for a period of 6 weeks.
(Karthikeyan M.,et al.)
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7. INSULIN DEFICIENCY DUE TO INSULIN ANTIBODIES
Principle and Rationale
Moloney and Coval(1995); Wright(1968)- A transient diabetic syndrome can be induced by injection of guinea pig anti-insulin
serum.
(Vogel,G.H.,et al.)
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Bovine insulin dissolved in acidified water (pH 3.0) at dose of 1mg/ml
Injected to male guinea pig (s.c).
Anti-insulin Sera is collected after two weeks of antigenic challenge.
PREPRATION OF ANTIBODY
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Adult albino rats are injected with 0.25-1.0 ml guniea pig anti insulin serum.
Increased of blood glucose level upto 300mg/dl.
The drug sample to be screened is given and glucose level is analysed to determine the
activity.
PROCEDURE
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A. SPONTANEOUSLY or GENETICALLY DERIVED DIABETIC ANIMAL
INVIVO MODELS FOR NIDDM
Spontaneously diabetic animals of type 2 diabetes may be obtained from the animals with one or several genetic mutations transmitted from generation to generation (e.g., ob/ob, db/db mice) or by selected from non-diabetic outbred animals by repeated breeding over several generation [e.g., (GK) rat, Tsumara Suzuki Obese Diabetes (TSOD) mouse].
(K. Srinivasan & P. Ramarao)
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1. BB rat2. WBN/KOB rat3.GOTO/KAKIZAKI
rat4. ZUKKAR fatty rat5. WDF/ TA-FA rat6. BHE rat
1. KK mice2. KK-Ay mice3. NOD mice4. Obese
hyperglycemic mice5. New Zealand obese
mice6. Transgenic mice
RATS MICE
Other Rat and Mice
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BB RAT• Bio Breeding Rat.• Insulin deficiency & insulitis due to
beta-cells destruction.• Nakhooda et al. 1978.
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• Classic model of hyperinsulinemic obesity.
• Obesity due autosomal recessive gene which develops at an early age.
• Diabetic phenotype develops due to lipotoxicity to beta cells.
ZUKKER DIABETIC FATTY RAT
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KK- Ay MICE • Derived from KK mice.• Carry yellow obese gene• Demonstrate the extra pancreatic action
of glimepirid (Satoh et al. 1994).• Also evaluate ciglitazone (Sohda et al.
1990) .
KK- MICE• Nakamura (1962), reported on a diabetic
strain of the KK mouse.• It is obese animale & showed
polyphagia and polyuria.• Mice at the age of seven months or older
showed glucosuria and blood sugar levels up to 320mg%.
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SAND RAT• Small rodent (gerbil).• Indigenous to desert region.• Access to food and water is limited.• Exhibit the genetic predisposition, if fed
with high calorie laboratory diet.
B. DIET / NUTRITION INDUCED DIABETIC ANIMAL
Some of the animal models exist in which diabetes is induced neither by chemicals nor by genetic defect. Sand rat, Tuco-Tuco and Spiny mouse are important models of nutritionally induced obesity and type 2 diabetes.
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• Two species of spiny mouse Acomys russates & cahirinus.
• Have congenital hyperplasia of pancreatic islets.
• When laboratory bred, A.cahirinus- weight gain on fat diet with hyperhlycemia.
SPINY MICE
(K. Srinivasan & P. Ramarao)
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C. NEONATAL STZ MODEL FOR NIDDM
Neonatal rats : STZ(90 mg /kg) i.p at birth or within the first five days
Rats showing fasting blood glucose level above 140 mg/ dl are considered diabetic.
Drug sample to be screened is administered by a suitable route and blood glucose level is
analyzed (K. Srinivasan & P. Ramarao)
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D. TRANSGENIC DIABETIC ANIMAL
Schaefer et al. 1994, described a transgenic mouse model of
chronic hyperglycemia.
Effect of single gene or mutation on diabetes can be
investigated in vivo.
Dissection of complex genetics of type 2 diabetes become
easier.
Highly sophisticated and costly procedure for the production
and maintenance.
Expensive for regular screening experiments.
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Purpose & Rationale: Was Described extensively by Ross (1927), the perfusion of rat liver from the Portal vein is the most widely used technique.
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EFFECT ON LIVER1. Isolated Rat Liver
INVITRO SCREENING OF HYPOGLYCEMICS
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2. Isolated Hepatocytes
Purpose & Rationale: Isolated Hepatocytes can be used
to study the effect of drugs on hepatic gluconeogenisis & other hepatic metabolic reactions such as ketone bodies formation and tricarboxylic acid cycle.
(Vogel,G.H.,et al.)
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• Male Wister Rats (200-250gm).• Anasthasized with hexobarbitol 150ml/kg.Animal
• Isolate & washed with 100ml heparinized saline solution.
• Oxygenated air tube connected to portal vein.• Perfusion is done & perfusate is collected.
Isolation & Treatment
• The sample for analysis are withdrawn by catheter & are evaluated for net glucose production.
Evaluation
Procedure
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Glucose is evaluated by glucose-oxidase method. And pyruvate, acetoacetate & lactate are evaluated by enzymatic method.
1. The cell suspension is preincubated.2.Substrats are added.
3. Test drug added.
1. Male Wister rat taken.2. Hepatocytes isolated by collagenase method
Procedure
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EFFECT ON MUSCLE CELL
Use of Isolated Diaphragm From Mice And Rats
Isolate diaphragm from rats and divide it in equal parts. Incubate it in kreb-henselit buffer with 5mM glucose, insulin or compound to be tested.
After 30 min, hemi diaphragm are blotted on tissue and frozen in liquid nitrogen. Powdered tissue is dissolved in 30% KOH. After freezing samples are centrifuged.
Glycogen Pallets are washed with 70% ethnol & lebelled C14 glycogen determined. Total glycogen is determined after hydrolysis to glucose
The total conc. dependence of glucose uptake & conversion into glycogen by insulin is determined.
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• Body Weight.
• HbA1c
• Lipid Profile.
• Serum Insulin Level.
• Histopathology of Pancreas & Liver.
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Evaluation
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References
1. Tripathi,K.D., “Essentials of medical pharmacology”, Jaypee
publications, 2006 (7): 235-236.
2. Vogel,G.H., “Drug Discovery And Evaluation (Pharmacological
Assays)”, Springer, 2002: 999-1044.
3. Gupta,S.K., “Drug Screening Method (Preclinical Evaluation of New
Drugs)”, Jaypee Brothers Medical Publishers Pvt Ltd., 2009: 602-610.
4. K. Srinivasan & P. Ramarao, “Animal models in type 2 diabetes
research: An overview”, Indian J Med Res 125, March 2007: 451-472.
5. S. Lenzen, “The mechanisms of alloxan- and streptozotocin-induced
diabetes”, Diabetologia, Springer-Verlag, 2008 (51): 216–226.
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5. Manish Pal Singh and Kamla Pathak, “Animal models for biological
screening of anti-diabetic drugs: An overview”, Pelagia Research
Library, European Journal of Experimental Biology, 2015, 5(5): 37-48.
6. Karthikeyan M, Balasubramanian T and Pawan Kumar, “In-vivo
Animal Models and In-vitro Techniques for Screening Antidiabetic
Activity”, J Develop Drugs 5, 2016 (2): 153-159.
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